Salmonella Typhi Haplotype 58 (H58) Biofilm Formation and Genetic Variation in Typhoid Fever Patients with Gallstones in an Endemic Setting in Kenya.

The causative agent of typhoid fever, Salmonella enterica serovar Typhi, is a human restricted pathogen. Human carriers, 90% of whom have gallstones in their gallbladder, continue to shed the pathogen after treatment. The genetic mechanisms involved in establishing the carrier state are poorly understood, but S. Typhi is thought to undergo specific genetic changes within the gallbladder as an adaptive mechanism. In the current study, we aimed to identify biofilm forming ability and the genetic differences in longitudinal clinical S. Typhi isolates from asymptomatic carriers with gallstones in Nairobi, Kenya. Whole genome sequences were analyzed from 22 S. Typhi isolates, 20 from stool and 2 from blood samples, all genotype 4.3.1 (H58). Nineteen strains were from four patients also diagnosed with gallstones, of whom, three had typhoid symptoms and continued to shed S. Typhi after treatment. All isolates had point mutations in the quinolone resistance determining region (QRDR) and only sub-lineage 4.3.1.2EA3 encoded multidrug resistance genes. There was no variation in antimicrobial resistance patterns among strains from the same patient/household. Non-multidrug resistant (MDR), isolates formed significantly stronger biofilms in vitro than the MDR isolates, p<0.001. A point mutation within the treB gene (treB A383T) was observed in strains isolated after clinical resolution from patients living in 75% of the households. Missense mutations in Vi capsular polysaccharide genes, tviE P263S was also observed in 18% of the isolates. This study provides insights into the role of typhoid carriage, biofilm formation, AMR genes and genetic variations in S. Typhi from asymptomatic carriers.

Typhoidal salmonella disease in Mukuru informal settlement, Nairobi Kenya; carriage, diversity, and antimicrobial resistant genes.

INTRODUCTION: Multiple studies have shown that typhoid fever is endemic in developing countries characterized by poor hygiene. A unique way of Salmonella Typhi (S.Typhi) pathogenicity is establishing a persistent, usually asymptomatic carrier state in some infected individuals who excrete large numbers of bacteria in faeces. This study aimed to determine the isolation rate of S.Typhi from blood and stool samples among cases and asymptomatic individuals in the Mukuru informal settlement and identify antibiotic resistance patterns within the same population. MATERIALS AND METHODS: We recruited 1014 outpatient participants presenting with typhoid-like symptoms in selected health centres in Nairobi, Kenya. Bacterial isolation was done on Xylose Lysine Deoxycholate agar (XLD) and Mac Conkey agar (Oxoid), followed by standard biochemical tests. Identification was done using API20E, and S.Typhi was confirmed by serotyping using polyvalent antisera 0-9 and monovalent antisera d. The Kirby-Bauer disc diffusion method was used to test the antimicrobial susceptibility of S.Typhi isolates, while Multi-Drug Resistant (MDR) strains were characterized using conventional PCR. RESULTS: Of 1014 participants, 54 (5%) tested positive for S.Typhi. Thirty-eight (70%) of the S.Typhi isolated were from stool samples, while sixteen (30%) were from blood. Three (0.2%) of the isolates were from asymptomatic carriers. Of the 54 S.Typhi isolates, 20 (37%) were MDR. Resistance to ciprofloxacin and nalidixic acid was 43% and 52%, respectively. Resistance to amoxicillin-clavulanic acid (a beta-lactam inhibitor) was 2%. The BlaTEM-1 gene was present in 19/20 (95%) MDR isolates. CONCLUSION: MDR S.Typhi is prevalent in Mukuru Informal settlement. The sharp increase in nalidixic acid resistance is an indication of reduced susceptibility to fluoroquinolones, which are currently the recommended drugs for the treatment of typhoid fever. This study highlights the need for effective antimicrobial stewardship and routine surveillance of antimicrobial resistance (AMR) to inform policy on the prevention and control of MDR Typhoid disease.

Multiple introductions of multidrug-resistant typhoid associated with acute infection and asymptomatic carriage, Kenya.

Background: Understanding the dynamics of infection and carriage of typhoid in endemic settings is critical to finding solutions to prevention and control. Methods: In a 3-year case-control study, we investigated typhoid among children aged <16 years (4670 febrile cases and 8549 age matched controls) living in an informal settlement, Nairobi, Kenya. Results: 148 S. Typhi isolates from cases and 95 from controls (stool culture) were identified; a carriage frequency of 1 %. Whole-genome sequencing showed 97% of cases and 88% of controls were genotype 4.3.1 (Haplotype 58), with the majority of each (76% and 88%) being multidrug-resistant strains in three sublineages of the H58 genotype (East Africa 1 (EA1), EA2, and EA3), with sequences from cases and carriers intermingled. Conclusions: The high rate of multidrug-resistant H58 S. Typhi, and the close phylogenetic relationships between cases and controls, provides evidence for the role of carriers as a reservoir for the community spread of typhoid in this setting. Funding: National Institutes of Health (R01AI099525); Wellcome Trust (106158/Z/14/Z); European Commission (TyphiNET No 845681); National Institute for Health Research (NIHR); Bill and Melinda Gates Foundation (OPP1175797).

High relatedness of invasive multi-drug resistant non-typhoidal Salmonella genotypes among patients and asymptomatic carriers in endemic informal settlements in Kenya.

Invasive Non-typhoidal Salmonella (iNTS) disease is a major public health challenge, especially in Sub-Saharan Africa (SSA). In Kenya, mortality rates are high (20-25%) unless prompt treatment is instituted. The most common serotypes are Salmonella enterica serotype Typhimurium (S. Typhimurium) and Salmonella enterica serotype Enteritidis (S. Enteritidis). In a 5 year case-control study in children residing in the Mukuru informal settlement in Nairobi, Kenya, a total of 4201 blood cultures from suspected iNTS cases and 6326 fecal samples from age-matched controls were studied. From the laboratory cultures we obtained a total of 133 S. Typhimurium isolates of which 83(62.4%) came from cases (53 blood and 30 fecal) and 50(37.6%) from controls (fecal). A total of 120 S. Enteritidis consisted of 70(58.3%) from cases (43 blood and 27 fecal) and 50(41.7%) from controls (fecal). The S. Typhimurium population fell into two distinct ST19 lineages constituting 36.1%, as well as ST313 lineage I (27.8%) and ST313 lineage II (36.1%) isolates. The S. Enteritidis isolates fell into the global epidemic lineage (46.6%), the Central/Eastern African lineage (30.5%), a novel Kenyan-specific lineage (12.2%) and a phylogenetically outlier lineage (10.7%). Detailed phylogenetic analysis revealed a high level of relatedness between NTS from blood and stool originating from cases and controls, indicating a common source pool. Multidrug resistance was common throughout, with 8.5% of such isolates resistant to extended spectrum beta lactams. The high rate of asymptomatic carriage in the population is a concern for transmission to vulnerable individuals and this group could be targeted for vaccination if an iNTS vaccine becomes available.

Factors associated with occurrence of salmonellosis among children living in Mukuru slum, an urban informal settlement in Kenya.

BACKGROUND: In Kenya, typhoid fever and invasive non-typhoidal salmonellosis present a huge burden of disease, especially in poor-resource settings where clean water supply and sanitation conditions are inadequate. The epidemiology of both diseases is poorly understood in terms of severity and risk factors. The aim of the study was to determine the disease burden and spatial distribution of salmonellosis, as well as socioeconomic and environmental risk factors for these infections, in a large informal settlement near the city of Nairobi, from 2013 to 2017. METHODS: Initially, a house-to-house baseline census of 150,000 population in Mukuru informal settlement was carried out and relevant socioeconomic, demographic, and healthcare utilization information was collected using structured questionnaires. Salmonella bacteria were cultured from the blood and faeces of children < 16 years of age who reported at three outpatient facilities with fever alone or fever and diarrhea. Tests of association between specific Salmonella serotypes and risk factors were conducted using Pearson Chi-Square (χ2) test. RESULTS: A total of 16,236 children were recruited into the study. The prevalence of bloodstream infections by Non-Typhoidal Salmonella (NTS), consisting of Salmonella Typhimurium/ Enteriditis, was 1.3%; Salmonella Typhi was 1.4%, and this was highest among children < 16 years of age. Occurrence of Salmonella Typhimurium/ Enteriditis was not significantly associated with rearing any domestic animals. Rearing chicken was significantly associated with high prevalence of S. Typhi (2.1%; p = 0.011). The proportion of children infected with Salmonella Typhimurium/ Enteriditis was significantly higher in households that used water pots as water storage containers compared to using water directly from the tap (0.6%). Use of pit latrines and open defecation were significant risk factors for S. Typhi infection (1.6%; p = 0.048). The proportion of Salmonella Typhimurium/ Enteriditis among children eating street food 4 or more times per week was higher compared to 1 to 2 times/week on average (1.1%; p = 0.032). CONCLUSION: Typhoidal and NTS are important causes of illness in children in Mukuru informal settlement, especially among children less than 16 years of age. Improving Water, Sanitation and Hygiene (WASH) including boiling water, breastfeeding, hand washing practices, and avoiding animal contact in domestic settings could contribute to reducing the risk of transmission of Salmonella disease from contaminated environments.

Multidrug-resistant Nontyphoidal Salmonella Hotspots as Targets for Vaccine Use in Management of Infections in Endemic Settings.

BACKGROUND: Salmonella infections cause a disproportionately high number of deaths in Africa, especially among poor urban populations. The increasing level of multidrug-resistant (MDR) infections is a major cause of concern in these settings where alternative effective treatment is unavailable. Other options for management of these infections must be sought. The knowledge of hotspots in endemic settings can help to prioritize management and control measures in Kenya and the region. METHODS: Using blood cultures, we investigated children presenting with fever of unknown origin for Salmonella infections. We performed antimicrobial susceptibility testing and whole genome sequencing to further characterize Salmonella isolates. Using Global Positioning System technologies, we mapped Salmonella isolates to households of patients in the study site and determined risk factors associated with high concentration of cases in particular sites. RESULTS: A total of 281 Salmonella species (149 from blood and 132 from fecal samples) from febrile children <5 years of age were studied. These consisted of 85 Salmonella Typhimurium, 58 Salmonella Enteritidis, 32 other nontyphoidal Salmonella (NTS) serotypes, and 126 Salmonella Typhi. The prevalence of MDR invasive NTS (iNTS) was 77.2%, with 15% resistant to ceftriaxone, a drug that is last-line treatment for iNTS and other severe gram-negative infections in Kenya. Invasive NTS and S. Typhi together mapped around common water vending points and close to sewer convergence points in the highly populated village. CONCLUSIONS: These hotspots could be targeted for management and control strategies, including a combined introduction of typhoid and iNTS vaccines, aimed at reducing transmission in these endemic settings.

Implementing a quality management system using good clinical laboratory practice guidelines at KEMRI-CMR to support medical research.

Background: Good Clinical Laboratory Practice (GCLP) is a standard that helps ensure the quality and reliability of research data through principles of Good Laboratory Practice (GLP) and Good Clinical Practice (GCP). The implementation of GCLP includes careful documentation of procedures, competencies and safety measures. Implementation of GCLP is influenced by existing resources and quality systems, thus laboratories in low- and middle-income countries may face additional challenges. Methods: This paper describes implementation of GCLP at the Kenya Medical Research Institute-Center for Microbiology Research (KEMRI-CMR) as part of a quality system to support medical research. This study employed assessment, twinning (institutional mentorship) model, conducting relevant training workshops and Kaizen 5S approaches to implement an effective quality management system using GCLP standard. This was achieved through a collaboration between the KEMRI/Wellcome Trust Research Programme (KWTRP) and KEMRI-CMR. The aim was compliance and continuous monitoring to meet international GCLP standards in a way that could be replicated in other research organizations. Results: Following a baseline assessment in March 2017, training, mentorship and a cycle of quality audit and corrective action using a Kaizen 5S approach (sorting, setting in order, shining, standardizing and sustaining) was established. Laboratory personnel were trained in writing standard operating procedures and analytical plans, microbiological techniques, and good documentation practice. Mid-term and exit assessments demonstrated significant declines in non-conformances across all GCLP elements. KEMRI-CMR achieved GCLP accreditation in May 2018 by Qualogy Ltd (UK). Conclusions: Involving all the laboratory personnel in implementation of quality management system processes is critical to success. An institutional mentorship (twinning) approach shows potential for future collaborations between accredited and non-accredited organizations to accelerate the implementation of high-quality management systems and continuous improvement.